The objective of this research proposal is to gain an understanding of the mechanisms underlying cell-type specific actions of activin on key targets of pituitary gonadotropes. Activins are pleiotropic regulators of diverse tissues and cellular functions, often acting through autocrine/paracrine mechanisms. They play a critical role in the pituitary to locally regulate gonadotrope function and promote the differential expression and secretion of the two gonadotropins, FSH and LH. Follistatins are secreted glycoproteins that function extracellularly to bind and modulate the local bioavailability of activin and other related ligands of the TGF- family through a feedback loop. In the pituitary, as in other tissues, the precise control of the activin/follistatin network helps maintain the functional integrity of target cells and prevents the development of pathologies. The dynamic control of activin/follistatin tone is achieved by the reciprocal actions of activin and follistatin and the self- modulating action of activin that exerts control on follistatin expression. Genetic models have substantiated the importance of follistatin by demonstrating that mice null for follistatin exhibit many embryonic defects and die shortly after birth while follistatin over-expression is associated with varying degrees of infertility. The follistatin gene is a transcriptional target of activin. In gonadotropes, this effect is mediated through the coordinated actions of the activin mediator, Smad3, and a member of the forkhead family of transcription factors, FoxL2. Functional and biochemical experiments have illustrated that FoxL2 is an obligatory partner of Smad3 for activin-dependent transcription of the follistatin gene in gonadotrope-derived cell types. FoxL2 is expressed in a limited number of tissues and human patients with FoxL2 deficiency display the Blepherophimosis-Ptosis- Epicanthus Inversus syndrome (BPES) associated with eyelid defects and premature ovarian failure in a sub- set of affected women. FoxL2 knockout mice exhibit a similar phenotype. FoxL2 is expressed in the pituitary of adult male and female mice and co-localizes with a-glycoprotein and FSH subunits. FoxL2 protein is also readily detectable in gonadotrope-derived cell lines and siRNA-mediated knockdown strategies have demonstrated that FoxL2 is a critical mediator of activin actions on key transcriptional targets that have thus far been identified such as follistatin, FSH and GnRH-R. By utilizing complimentary in vitro and in vivo models, the proposed studies of this application aim to provide a better understanding of the central role of FoxL2 in coordinating the Smad3-dependent activin program of gonadotropes, elucidate the mechanism underlying the partnership between FoxL2 and Smad3 and determine how FoxL2 function contributes to the local control of the activin/follistatin network of the pituitary, mechanisms that might prove to be relevant to other FoxL2-expressing tissues. Understanding the mechanism underlying FoxL2 action could illuminate strategies for the differential control of Smad3 targets and identify novel molecular targets with relevance to reproductive and other endocrine disorders. PUBLIC HEALTH RELEVANCE: Activins are ubiquitous growth factors known to regulate many cellular processes, including differential FSH expression and secretion from pituitary gonadotropes. Activins control their own bioactivity in part by exerting positive effects on the expression of activin-binding proteins known as follistatins, which are also ubiquitous and often co-localize with activins. Through a better understanding of how activin regulates the expression of follistatin and other key targets in gonadotropes, these studies will facilitate the identification of novel targets that have the potential to selectively target the tissue-specific actions of activin relevant to reproductive and/or other endocrine pathologies.